Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs

Opiate drugs such as morphine and heroin are among the most effective analgesics known. Prolonged or repeated administration of opiates produces adaptive changes in the nervous system that lead to reduced drug potency or efficacy (tolerance), as well as physiological withdrawal symptoms and behavioral manifestations such as craving when drug use is terminated (dependence). These adaptations limit the therapeutic utility of opiate drugs, particularly in the treatment of chronically painful conditions, and are thought to contribute to the highly addictive nature of opiates. For many years it has been proposed that physiological tolerance to opiate drugs is associated with a modification of the number or functional activity of opioid receptors in specific neurons. We now understand certain mechanisms of opioid receptor desensitization and endocytosis in considerable detail. However, the functional roles that these mechanisms play in the complex physiological adaptation of the intact nervous system to opiates are only beginning to be explored.     

Mechanism of development of tolerance to injected morphine by guinea pig ileum.

Injection of a large dose of morphine into a guinea pig results in a block of electrically-induced contractions of the ileum $\text{in vitro}$. A similar dose is almost ineffective in guinea pigs given morphine chronically. The time course for development of this tolerance has been determined in guinea pigs injected twice daily with morphine 100 mg/kg and challenged on various days with 750 mg/kg of the drug. Animals similarly injected but not challenged served as controls. The inhibitory effect of the challenging dose on electrical stimulation of longitudinal muscle decreased with successive days of morphine administration; by the 10th day there was almost complete tolerance to the challenging dose. Sensitivity of the tissues of chronically morphinized unchallenged controls towards acetylcholine, serotonin, histamine and norepinephrine was essentially the same as that of naive animals. The potency of morphine $\text{in vitro}$ in blocking electrical stimulation was also unchanged by chronic morphine administration in the above manner. Thus tolerance to injected morphine cannot be explained by reduced affinity of the drug for the opiate receptor. Tissues of chronically morphinized animals gave a contracture with naloxone, the extent of the contracture increasing with time of drug administration. This naloxone effect is attributed to displacement of morphine from a new opiate receptor site induced during morphine administration. It is suggested that this new receptor is involved in tolerance to injected morphine as well as some aspects of the withdrawal syndrome.     

Depression of Mesolimbic Dopamine Transmission and Sensitization to Morphine During Opiate Abstinence

To investigate the role of mesolimbic dopamine (DA) in the mechanism of drug dependence, extracellular DA was monitored by transcerebral dialysis in the caudal nucleus accumbens under basal conditions and after challenge with morphine (5 mg/kg s.c.) in control rats and in rats made dependent on and then deprived of morphine. Withdrawal from morphine resulted in a marked reduction of extracellular DA concentrations from control values at 1, 2, 3, and 5 days of withdrawal. After 7 days of withdrawal, DA output was less, but still significantly, reduced. Challenge with morphine resulted in stimulation of DA output in controls (maximum, 35%), no effect on the first day of withdrawal, and stimulation similar to controls' on days 2 and 7 of withdrawal. On day 5 and, particularly, on day 3 of withdrawal, morphine-induced stimulation of DA output was markedly potentiated (maximum, 100 and 160%, respectively). Changes in the sensitivity of DA transmission to morphine challenge were associated with changes in the behavioral stimulant effects of morphine, with tolerance on day 1 and marked sensitization on days 3 and 5 but also on day 7, when morphine-induced stimulation of transmission was no longer potentiated. The results indicate that repeated morphine administration induces a state of dependence in DA neurons and a short-lasting tolerance followed by an increased sensitivity to its stimulant effects on DA transmission. These changes might play an important role in the development of opiate addiction and in the maintenance of opiate self-administration in dependent subjects.     

The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in 3H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine.     

Role of endothelin (ETA) receptors in neonatal morphine withdrawal

We have previously demonstrated role of central endothelin (ET) receptors in neonatal morphine tolerance. The present study was conducted to investigate involvement of central ET receptors in neonatal rat morphine withdrawal. The aim was to determine activation of G-proteins coupled to opioid and ET receptors by morphine and ET ligands in neonatal rat brains during morphine withdrawal. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets over 7 days. Withdrawal was induced on day 8 by removal of pellets. Rat pups were delivered by cesarean section 24 h after pellet removal. G-protein stimulation induced by morphine; ET-1; ETA receptor antagonist, BMS182874; and ETB receptor agonist, IRL1620, was determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTPgammaS binding assay. Morphine-induced maximal stimulation of G-protein in morphine withdrawal group (83.60%) was significantly higher compared to placebo control group (66.81%). EC50 value for ET-1-induced G-protein stimulation during morphine withdrawal (170.60 nM) was higher than control (62.5 nM). BMS182874, did not stimulate GTP binding in control but significantly increased maximal stimulation of G-proteins in morphine withdrawal (86.07%, EC50 = 31.25 nM). IRL1620-induced stimulation of G-proteins was similar in control and morphine withdrawal. The present findings indicate involvement of central ETA receptors in neonatal morphine withdrawal.     

Serum IL-10 involved in morphine tolerance development during adjuvant-induced arthritis

Opioid receptors play an important role in modulation of hyperalgesia in inflamed tissues, but chronic morphine application induces such side effects as tolerance. There is near communications between cytokines and mu opioid receptor expression. This study was aimed to assess the role of serum IL-10 in morphine tolerance development during adjuvant-induced arthritis. Adjuvant arthritis (AA) was induced on day 0 by single injection of Complete Freund’s Adjuvant (CFA) into the rats’ hindpaw. Hyperalgesia, edema, and spinal mu opioid receptor (mOR) variations were assessed on 0, 7, 14, and 21 days of the study. For assessment of the morphine tolerance development, morphine effective dose (4 mg/kg) was administered from the 14th day after CFA injection and continued until the morphine post-dose paw withdrawal latency (PWL); it did not significantly differ from the baseline. For assessment of the effects of IL-10 on tolerance induction, a neutralizing dose (ND50) of anti-IL-10 was administered daily during different stages of the study. AA induction in the right hindpaw of rats resulted in unilateral inflammation and hyperalgesia within 21 days of the study. Anti-IL-10 antibody administration in the AA rats induced marked elevation of hyperalgesia compared to the AA control group. Our data also indicated that morphine effective anti-hyperalgesic dose significantly decreased in the AA rats compared to the control group, which this symptom was aligned with spinal mu opioid receptor (mOR) expression increase during AA. Moreover, there was a significant difference in morphine tolerance induction between the AA and control rats, and our results also demonstrated that IL-10 played an important role in tolerance-induction process. It can be concluded that morphine tolerance slowly progressed when administered morphine effective dose was reduced during AA chronic inflammation. On the other hand, it seems that increased level of serum IL-10 may affect morphine tolerance development during inflammation.     

Involvement of central endothelin receptors in neonatal morphine withdrawal

The involvement of central endothelin (ET) receptors in neonatal morphine tolerance has been demonstrated. The present study investigates the role of central ET receptors in morphine withdrawal in neonatal rats. The aim was to determine whether activation of G-proteins coupled to opioid and ET receptors by morphine and various ET receptor modulators is affected during morphine withdrawal in neonatal rats. Pregnant female rats were rendered tolerant to morphine by chronic exposure to morphine pellets during 7 days. On Day 8, pellets were removed and rats were allowed to undergo withdrawal for 24 hrs. Rat pups were delivered by cesarean section. G-protein stimulation induced by morphine; ET-1; the ET(A) receptor antagonist, BMS182874; and the ET(B) receptor agonist, IRL1620, were determined in the brain of neonatal rats undergoing morphine withdrawal by [35S]GTPgammaS binding assay. Morphine produced higher (P < 0.05) maximal stimulation of G-protein in the morphine-withdrawal group (83.60%) compared with the placebo group (66.81%). ET-1-induced G-protein stimulation was also altered, and the median effective concentration (EC50) during morphine withdrawal (170.60 nM) was significantly higher than placebo (62.5 nM; P< 0.05). The maximal stimulation induced by the ET(A) receptor antagonist, BMS182874, in the morphine-withdrawal group (86.07%; EC50 = 31.25 nM) was significantly higher than in the placebo group (EC50 > 1000 nM). The ET(B) agonist, IRL1620, induced G-protein stimulation was similar in placebo (73.43%, EC50 = 13.26 nM) and morphine-withdrawal groups (75.08%, EC(50) = 11.70 nM), respectively. To our knowledge, this is the first report indicating involvement of central ET(A) receptors in neonatal morphine withdrawal.     

The effects of pituitary adenylate cyclase-activating polypeptide on acute and chronic morphine actions in mice

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on pain sensitivity, on morphine analgesia, on morphine tolerance and withdrawal were investigated in mice. The heat-radiant tail-flick test was used to assess antinociceptive threshold. Intracerebroventricular (i.c.v.) administration of PACAP alone had no effect on pain sensitivity but in a dose of 500 ng, it significantly diminished the analgesic effect of a single dose of morphine (2.25 mg/kg, s.c.). PACAP (500 ng, i.c.v.) significantly increased the chronic tolerance to morphine and enhanced the naloxone (1 mg/kg, s.c.)-precipitated withdrawal jumping. Theophylline (1 mg/kg, i.p.) pretreatment significantly enhanced the effect of PACAP on morphine analgesia but the effects of PACAP on tolerance and withdrawal were unaffected upon theophylline administration. On the grounds of our previous studies with vasoactive intestinal polypeptide (VIP), it appears that different receptors are involved in the effects of PACAP in acute and chronic morphine actions. Our results indicate that PACAP-induced actions likely participate in acute and chronic effects of morphine and suggest a potential role of PACAP in opioid analgesia, tolerance and withdrawal.     

Role of delta opioid receptors and their ligands in the development of adaptive heart protection against arrhythmogenesis

It has been found that stimulation of delta-1 opioid receptors by intravenous administration of DPDPE (0.5 mg/kg) decreases the incidence of ischemic and reperfusion-induced arrhythmias and also increases myocardial tolerance to the arrhythmogenic action of epinephrine in rats. Pretreatment with a selective delta-2 agonist, DSLET, had no antiarrhythmic effect. The inhibition of the enzymatic breakdown of endogenous enkephalins by intravenous administration of acetorphan decreased the incidence of epinephrine-induced arrhythmias. Pretreatment with a selective delta opioid receptor antagonist, ICI-174.868, completely abolished this antiarrhythmic effect. Adaptation of rats to repeated immobilization stress during 12 days increased myocardial tolerance to the arrhythmogenic action of coronary artery occlusion (10 min) and reperfusion (10 min). Pretreatment with a selective delta opioid receptor antagonist, TIPP(Psy), did not abolish the antiarrhythmic effect of adaptation to immobilization stress. It seems that endogenous agonists of delta opioid receptors are not involved in the antiarrhythmic effect resulting from adaptation to stress.     

Multiple actions of spinophilin regulate mu opioid receptor function

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

Background

Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated.

Results

Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids.

Conclusion

In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.     

Alpha-adrenergic agents have little effect during air embolism lung injury in awake sheep

Since it is not clear whether alpha-adrenergic receptors can modulate lung microvascular liquid and protein leakiness, we studied the effects of alpha-adrenergic receptor stimulation or blockade on lung filtration under base-line conditions and during the acute lung injury caused by a 4-h infusion of venous air emboli in six unanesthetized, chronically instrumented sheep with lung lymph fistulas. During the experiments we continuously infused the alpha-adrenergic receptor agonist phenylephrine hydrochloride (1.0 microgram X kg-1 X min-1 iv) or the alpha-adrenergic receptor antagonist phentolamine mesylate (1.0 mg X kg-1 X min-1 iv), and we measured pulmonary vascular pressures, cardiac output, lung lymph flow, and the lymph-to-plasma protein concentration ratio. During air embolism, alpha-receptor stimulation increased pulmonary vascular resistance and decreased lung lymph flow by 25%; alpha-receptor blockade had the opposite effects. During recovery, neither agent significantly affected pulmonary hemodynamics or lymph flow. Our results indicate that alpha-adrenergic receptors are active during air embolism and modulate pulmonary filtration by causing arteriolar constriction, which reduces the surface area or the perfusion pressure in the pulmonary microvascular bed. They may also affect venous smooth muscle tone. We found no evidence that alpha-adrenergic receptors modulate lung microvascular liquid or protein leakiness directly.     

Angiotensin II mediates uterine vasoconstriction through alpha-stimulation

Intravenous angiotensin II (ANG II) increases uterine vascular resistance (UVR), whereas uterine intra-arterial infusions do not. Type 2 ANG II (AT(2)) receptors predominate in uterine vascular smooth muscle; this may reflect involvement of systemic type 1 ANG II (AT(1)) receptor-mediated alpha-adrenergic activation. To examine this, we compared systemic pressor and UVR responses to intravenous phenylephrine and ANG II without and with systemic or uterine alpha-receptor blockade and in the absence or presence of AT(1) receptor blockade in pregnant and nonpregnant ewes. Systemic alpha-receptor blockade inhibited phenylephrine-mediated increases in mean arterial pressure (MAP) and UVR, whereas uterine alpha-receptor blockade alone did not alter pressor responses and resulted in proportionate increases in UVR and MAP. Although neither systemic nor uterine alpha-receptor blockade affected ANG II-mediated pressor responses, UVR responses decreased >65% and also were proportionate to increases in MAP. Systemic AT(1) receptor blockade inhibited all responses to intravenous ANG II. In contrast, uterine AT(1) receptor blockade + systemic alpha-receptor blockade resulted in persistent proportionate increases in MAP and UVR. Uterine AT(2) receptor blockade had no effects. We have shown that ANG II-mediated pressor responses reflect activation of systemic vascular AT(1) receptors, whereas increases in UVR reflect AT(1) receptor-mediated release of an alpha-agonist and uterine autoregulatory responses.     

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.     

Acute cross-tolerance to opioids in heroin delta-opioid-responding Swiss Webster mice

It is generally thought that the mu receptor actions of metabolites, 6-monoacetylmorphine (6MAM) and morphine, account for the pharmacological actions of heroin. However, upon intracerebroventricular (i.c.v.) administration in Swiss Webster mice, heroin and 6MAM act on delta receptors while morphine acts on mu receptors. Swiss Webster mice made tolerant to subcutaneous (s.c.) morphine by morphine pellet were not cross-tolerant to s.c. heroin (at 20 min in the tail flick test). Now, opioids were given in combination, s.c. (6.5 h) and i.c.v. (3 h) preceding testing the challenging agonist i.c.v. (at 10 min in the tail flick test). The combination (s.c. + i.c.v.) morphine pretreatment induced tolerance to the mu action of morphine but no cross-tolerance to the delta action of heroin, 6MAM and DPDPE and explained why morphine pelleting did not produce cross-tolerance to s.c. heroin above. Heroin plus heroin produced tolerance to delta agonists but not to mu agonists. Surprisingly, all combinations of morphine with the delta agonists produced tolerance to morphine which now acted through delta receptors (inhibited by i.c.v. naltrindole), an unusual change in receptor selectivity for morphine.     

Effects of prenatal cocaine, morphine, or both on postnatal opioid (mu) receptor development

We studied the effects of prenatal cocaine and morphine given separately and in combination on the (1) postnatal brain mu-opioid receptor development and (2) interaction of dopamine with mu receptors. Pregnant rats received single daily intraperitoneal (I.P.) injections of saline, cocaine (20 mg/kg), morphine (2 mg/kg), or the combination of both drugs from day 13 to day 20 of gestation. Postnatal days (P) 1, 7, 14, and 28, whole brains were analyzed for opioid receptor binding and mu mRNA. Prenatal cocaine administered by itself had no significant effect on the ontogeny of brain mu receptors on all the days studied when compared to controls. The morphine-treated group showed a significant increase in mu receptor binding on P1 and P7. Exposure to both cocaine and morphine showed a significant increase in mu receptor density on P1 and P7. In addition, there was also a significant increase in MOR mRNA in both the morphine alone and combination groups. Pretreatment with dopamine D2 receptor antagonist (sulpiride, 20 mg/kg) prior to drug administration showed decreased mu receptor binding on P1 and P7. These results suggest that prenatal exposure to morphine or a combination of cocaine and morphine significantly increases mu receptor density. By P14, mu-opioid receptor binding was no longer different than the control. This may suggest that the effect on receptor may be short-lived and that other key intracellular events may be activated to mediate the long-term effects. Also, the data show that dopaminergic mechanisms are (or opioid-dopamine interaction is) involved in the effects of morphine alone or morphine in combination with cocaine on mu receptor regulation.     

Modulation of acute morphine tolerance by corticotropin-releasing factor and dynorphin A in the mouse spinal cord.

Previously, we have demonstrated that intrathecally (i.t.) administered corticotropin-releasing factor (CRF) in mice produces stimulus-specific antinociception and modulation of morphine-induced antinociception by mechanisms involving spinal kappa opioid receptors. Recently, we also have found that CRF releases immunoreactive dynorphin A, a putative endogenous kappa opioid receptor agonist, from superfused mice spinal cords in vitro. Dynorphin A administered intracerebroventricularlly (i.c.v.) to mice has been shown to modulate the expression of morphine tolerance. In the present study, the possible modulatory effects of i.t. administered CRF as well as dynorphin A on morphine tolerance were studied in an acute tolerance model. Subcutaneous administration of 100 mg/kg of morphine sulfate (MS) to mice caused an acute tolerance to morphine-induced antinociception. The antinociceptive ED50 of MS was increased from 4.4 mg/kg (naive mice) to 17.9 mg/kg (4 hours after the injection of 100 mg/kg MS). To study the modulatory effects of spinally administered CRF and dynorphin A on the expression of morphine tolerance, CRF and dynorphin A were injected i.t. at 15 min and 5 min, respectively, before testing the tolerant mice by the tail-flick assay. The antinociceptive ED50 of MS in tolerant mice was decreased to 8.8 mg/kg and 7.1 mg/kg, respectively, after i.t. administration of CRF (0.1 nmol) and dynorphin A (0.2 nmol). In contrast, 0.5 nmol of alpha-helical CRF (9-41), a CRF antagonist and 0.4 nmol of norbinaltorphimine, a highly selective kappa opioid receptor antagonist, when administered i.t. at 15 min before the tail-flick test in tolerant mice, increased the antinociceptive ED50 of MS to 56.6 mg/kg and 88.8 mg/kg, respectively. These data confirmed the modulatory effect of dynorphin A on morphine tolerance and suggested that CRF, which releases dynorphin A in several central nervous system regions, also plays a modulatory role in the expression of morphine tolerance.     

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

PDGF alpha- and beta-receptors activate unique and common signal transduction pathways.

We have examined the signal transduction pathways of the PDGF alpha- and beta-receptors, in order to characterize the specificity of each receptor type in the signaling. Porcine aortic endothelial cell lines expressing equal levels of either PDGF alpha- or beta-receptors were established. The alpha- and beta-receptor cells responded mitogenically to stimulation with the proper PDGF isoforms. Three aspects of actin reorganization were examined after ligand stimulation: loss of stress fibres, appearance of edge ruffles and formation of circular membrane ruffles. The beta-receptor cells showed a response to ligand stimulation which included all three features. The alpha-receptor cells exhibited edge ruffles and loss of stress fibres, but circular ruffles could not be found in several independent alpha-receptor cell lines. The beta-receptor cells, but not the alpha-receptor cells, were able to migrate chemotactically towards a concentration gradient of ligand. The molecular basis for the differences in signalling were explored by comparing the pattern of increased phosphorylation of potential substrates for the alpha- and beta-receptors in [32P]orthophosphate labelled intact cells and using an in vitro kinase assay. Certain of the observed substrates were common for the two receptors, whereas others were specific for either one. We conclude that certain of the known PDGF induced cellular effects are transduced only by the beta-receptor; the presence of alpha-receptor-specific substrates suggests that there are also alpha-receptor-specific signals, which have yet to be identified.